Aflatoxins are a group of highly toxic metabolites mainly produced by Aspergillusflavus and Aspergillus parasiticus. Aflatoxins are one category of most powerful carcinogenic substances ever discovered. 20 kinds of aflatoxins have now been found, mainly including aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2) and M1 (AFM1), etc. Among them, AFB1 is the most toxic. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of AFB1. When mammals ingest a feed contaminated by AFB1, AFB1 would be hydroxylated in vivo and secreted into milk. In general, after the animals ingesting a food contaminated by AFB1, the discharge amount of AFM1 is 1%-3% of the intake amount of AFB1. A large number of researchers have conducted deep research on the toxicity and carcinogenicity of AFM1, and the research results motivated International Agency for Research on Cancer to change the carcinogenic rank of AFM1 from the category II carcinogenic substance to the category I carcinogenic substance. AFM1 is stable in property and almost completely impossible to be destroyed even if it is subjected to pasteurization. AFM1 is present in many dairy products. Since dairy products are the main source of infant foods, the problems about the AFM1 contamination have attracted worldwide attention and the amount of AFM1 is strictly limited in those dairy products. China belongs to heavily contaminated areas of aflatoxins, and therefore it is of important significance to intensify the detection especially the rapid detection of AFM1 in milk and dairy products to timely understand and grasp the health information about the milk and dairy products for ensuring the safety of food consumption in China.
The existing detection methods for aflatoxins include thin-layer chromatography, precision instrument analysis method and immunological analysis method. Among them, thin-layer chromatography is the most common detection method used for aflatoxins very early. Thin-layer chromatography does not need special instruments and equipment and can be carried out in ordinary laboratories, but it has the problems of large reagent consumption, tedious operation, severe interference by other components, poor accuracy, incapability of accurate quantification, great harm to experimenters and surrounding environment, and inapplicability to in-field rapid detection. The precision instrument analysis method mainly includes fluorospectrophotometry and high performance liquid chromatography. These methods have high sensitivity and good accuracy, but they require high purity of aflatoxin samples, require tedious traditional sample pretreatment processes such as liquid-liquid extraction, solid phase extraction, solid-phase microextraction, and have low specificities. Therefore, the establishment of rapid and effective sample pretreatment techniques has become the principle and bottleneck problem in the detection and analysis of aflatoxins. An immunoaffinity column is a novel highly efficient sample pretreatment means, which is based on reversible specific binding between antigens and antibodies to realize the enrichment and purification of the target substance in a complex sample. The combination of the immunoaffinity column with liquid phase chromatographic analysis, fluorescence tachometer and the ELISA method can be widely used in the detection of aflatoxins in agricultural products and foods.
Currently, the preparation of immunoaffinity column for aflatoxins is implemented by coupling traditional antibodies (polyclonal antibodies or monoclonal antibodies) with an agarose gel or silica gel microspheres. Since the activities of the traditional antibodies degrade rapidly during use, it is a technical problem that the repeatable times for use of the existing immunoaffinity column on the market are relatively less. A nanobody is a heavy chain antibody naturally found in camelidae. Comparing with traditional antibody, the nanobody has advantages such as good stability, tolerance to high temperature, tolerance to acids and bases, and tolerance to organic reagents, and can be used in preparing an immunoaffinity column to prolong the shelf life of the immunoaffinity column. Further, nanobody can be produced through genetic engineering means, and thus the production cost of nanobody is low, and the production of nanobody is easy. Therefore, nanobody is more advantageous than conventional antibody in the preparation of an immunoaffinity column. Currently, there is still no report related to the immunosorbent and immunoaffinity column for aflatoxin nanobodies.